The structural basis of the activation and inhibition of DSR2 NADase by phage proteins

DSR2, a Sir2 domain-containing protein, protects bacteria from phage infection by hydrolyzing NAD+. The enzymatic activity of DSR2 is triggered by the SPR phage tail tube protein (TTP), while suppressed by the SPbeta phage-encoded DSAD1 protein, enabling phages to evade the host defense. However, the molecular mechanisms of activation and inhibition of DSR2 remain elusive. Here, we report the cryo-EM structures of apo DSR2, DSR2-TTP-NAD+ and DSR2-DSAD1 complexes. DSR2 assembles into a head-to-head tetramer mediated by its Sir2 domain. The C-terminal helical regions of DSR2 constitute four partner-binding cavities with opened and closed conformation. Two TTP molecules bind to two of the four C-terminal cavities, inducing conformational change of Sir2 domain to activate DSR2. Furthermore, DSAD1 competes with the activator for binding to the C-terminal cavity of DSR2, effectively suppressing its enzymatic activity. Our results provide the mechanistic insights into the DSR2-mediated anti-phage defense system and DSAD1-dependent phage immune evasion.

1.The authors stated that the DSR2 H171A lost its ability to bind NAD, based on the fact that no NAD density was observed in the sample of DSR2 H171A with extra NAD.This conclusion is too arbitrary, which needs to be proved by measuring their affinity.2. Then sentence "The HTH domain folds into two HTH motifs and a β hairpin between.' is incomplete.3. Structural comparison of DSR2 and ThsA, Y134 and D135 are supposed to be important for NAD coordination, this conclusion needs to be proved by the biochemical experiments.4. In the provided PDB (6LHX), the structure is the apo form ThsA, it is hard to identified the residues for NAD binding.The structure of NAD bound Sir2 in the Sir2-APAZ/pAgo was reported (PMID: 38200015), maybe the comparison of Sir2 in DSR2 and pAgo would be helpful.5.In figure 2f and extended figure 1a, the results of gel filtration profile of DSR2 are inconsistent.The peak is 9 ml and 50 ml, please explain.Besides, it is better to use Analytical ultracentrifugation to analysis the effect of the mutation on its oligomerization.Why R86E and Y260E (they are both in the interface 4 ) are eluted differently?6.The author state that the oligomeric DSR2 lacks NAD hydrolysis activity, so why they used DSR2 H171A mutant to collect the cryo-EM data?If the DSR2 alone is inactivated, how the DSAD1 inhibits its NADase activity?Maybe DSAD1 and the phage tail protein (the activator) competitive bind DSR2?So, the author needs to test the binding affinity of DSR2 and DASD1, and phage tail.
Reviewer #3: Remarks to the Author: Bacteria and archaea use an expanded set of antiviral defenses to protect against infection by phage.One such defense system, DSR2, uses an NAD+ depletion strategy through the activity of a SIR2 domain.In this manuscript, Wang and Xu et al. use cryo-EM to determine the 3D structure of a tetrameric DSR2 complex providing the first structural information regarding this defense system.The authors also reveal the structure of DSR2 in complex with the virally encoded antidefense protein DSAD1 which was previously shown to inhibit DSR2 enzymatic activity.These structures suggest a mechanism for anti-defense by DSAD1 which likely locks DSR2 in a conformation which is not conducive to NAD+ binding and catalysis.
The results of this manuscript are clearly presented.The structural information provided in this manuscript adds to our understanding of NAD+ depleting prokaryotic defense systems which operate through oligomerization.The manuscript, while well organized, lacks data which would shore up some of the conclusions and make it overall a more comprehensive story.Importantly, NADase assays are absent in the current study but would be essential to explain how the oligomers observed in cryo-EM maps may be operating.

Suggested revisions:
• Missing word in introduction-"While in bacteria, Sir2 domain proteins recently identified to act as NADases to hydrolyze the bacterial cellular NAD+ to against phage infection."-Missing a word between to and against.Perhaps "protect"?• The fact that NAD+ was not observed in the binding pocket of DSR2 may be due to the mutation but it is more than likely that NAD+ cannot bind until DSR2 is conformationally triggered by phage tail tube protein.This alternative interpretation comes in some form much later in the discussion but may be worth stating much earlier.
• Typo TshA -> ThsA "To elucidate the mechanism of NAD+ binding, we conducted a detailed analysis of the overlaid structures of DSR2 and TshA." • Can the authors more accurately/quantitatively describe the oligomeric state of the tetramer interface mutants?"The gel filtration profiles of the DSR2 mutant proteins exhibited slower migration compared to the wild-type protein, indicating disruption of DSR2 tetramerization (Fig. 2f)."If molecular weight standards were run this could indicate if the shifted peaks are representative of monomer or dimeric complexes.
• While the subject of the manuscript is the inactivation mechanism of DSAD1 on DSR2 as determined by cryo-EM, there is no NADase data presented showing any inactivation.It might be beneficial to understanding the mechanism if some NADase measurements could be made.At minimum, the impact of tetramer and dimer interface mutants on NADase activity should be explored to highlight the importance of these complexes on activity but also the impact of DSAD1 mutants on NADase inactivation could be explored to further emphasize the importance of these interactions.
• Unless the authors can show it directly using mutagenesis, binding affinity measurements, and/or cryo-EM, the references to competition of binding to DSR2 by DSAD1 and tail tube should be removed.The model as it stands is incomplete without evidence of direct competition.The analysis should reflect this uncertainty-DSAD1 may bind to a completely separate site relative to the binding site for tail tube and therefore binding would not be considered competitive.For example, there is no evidence to suggest that DSAD1 dislodges the tail tube as seemingly presented in Fig. 5d.

Point-to-point response to reviewers' comments
Manuscript ID: NCOMMS-24-06231-T Previous Title: The structural basis of DSAD1-DSR2 mediated phage immune evasion Current Title: The structural basis of the activation and inhibition of DSR2 NADase by phage proteins We express our gratitude to the reviewers for their valuable and constructive comments.We have addressed their comments raised during the previous round of review, resulting in significant improvements to the manuscript.Please find our point-by-point responses to the reviewers below.The reviewer's comments are presented in black text, while our responses are highlighted in blue.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): DSR2 forms a tetrameric structure with considerable structural flexibility.This is an inactive form of the protein, which requires binding to the phage tail tube protein for activation.DSAD1 binds this complex in a 2:4 ratio.Key predicted interface residues are tested by sdm, confirming changes in affinity (see point 2 below).
The authors suggest DSAD1 binding induces inactivating structural changes in DSR2 and present a model based on their data, whereby DSAD1 displaces the phage tail-tube protein to inactivate DSR2 (Figure 5d)see point 3 below.
Overall, there is some interesting structural data here, but the lack of supporting biochemical data, particularly activity data, reduces confidence in the conclusions drawn.

Response:
We thank the reviewer for the excellent summary of our work and for the positive comments.
Major points.
1.The bulk of the experimental data in this manuscript consists of cryoEM data.This should be carefully checked by an expert reviewer.

Response:
We carefully processed and presented our cryoEM results and the data was evaluated by other reviewers.
2. The MBP-pull down experiment used to quantify the affinity between DSR2 and DSAD1 wt and variants does not yield data that allow a firm conclusion about the importance of any particular residue for binding.It would be far better to assay the activity of DSR2 in the presence of the various mutants.The authors mention the use of a well-established NADase assay for DSR2 but note "data not shown", which is not really acceptable.The authors seem to have everything they need to do these assays, even if the tail-tube protein is not suitable for structural analyses.
Response: This is a great suggestion.We have optimized our experiment system and carried out the in vitro NAD + hydrolase assays to analyze the enzymatic activities of WT DSR2 and its mutants in the presence of the tail tube protein (TTP) (Updated Figs.1b, j and Fig. 2f).TTP is essential for the NAD + hydrolase activity of DSR, which is suppressed by the wildtype DSAD1 but less by its mutants, supporting our obsevation in the complex structure (Updated Figs.5f and 6a, b).
3. The authors have a structure for inactive DSAD1 and another for DSAD1 bound to DSR2.They postulate that DSAD1 displaces the tail tube protein from DSR2 to inactivate the protein.This poses a number of problems.Firstly, in the absence of an activated structure, it is impossible to say whether DSAD1 causes inactivating structural rearrangement in the enzymatic domain of DSR2 along the lines shown in figure 5. Secondly, it is not ruled out, and seems much more likely, that DSAD1 binds DSR2 and prevents binding of the tail tube protein, rather than displacing it as shown in figure 5d.After all, DSAD1 is likely expressed very early in infection and tail tube protein expressed later.To test this, activity studies with differential order of addition of components could be carried out to see whether tail tube protein is displaced by DSAD1 or just prevented from binding to DSR2.

Response:
We appreciate the reviewer's insightful suggestion.In the revised manuscript, we successfully determined the cryo-EM structure of the DSR2-TTP-NAD + complex (Updated Fig. 3).This new structure provides insights into the interactions between DSR2 and TTP, revealing that TTP occupies the same pocket on DSR2 as DSAD1, indicating a competitive binding scenario between DSAD1 and TTP.To investigate the mechanism by which DSAD1 affects the binding of the TTP to DSR2, we performed the NADase assay by introducing DSAD1 and TTP to DSR2 in different orders.The activity studies demonstrated that DSAD1 effectively suppresses the enzymatic activity of DSR2 triggered by TTP, regardless of whether DSR2 encounters the TTP or DSAD1 first (Updated Figs.6a-b).However, TTP can not obviously activate DSR2 in the presence of DSAD1.Additionally, the subsequent pull-down assay revealed that DSAD1 significantly inhibits the binding of TTP to DSR2, even when DSR2 initially encounters TTP (Updated Fig. 6c).
These new results suggest that DSAD1 is a potent competitor of TTP for DSR2 binding, although they may not occur in cells.
Reviewer #2 (Remarks to the Author): In this manuscript, Wang et al reports the inhibitory mechanism of the DSR2 NADase activity by DSAD1, using the cryo-EM method.However, I still have some comments: Response: We thank the reviewer for the constructive comments.
1.The authors stated that the DSR2 H171A lost its ability to bind NAD, based on the fact that no NAD density was observed in the sample of DSR2 H171A with extra NAD.This conclusion is too arbitrary, which needs to be proved by measuring their affinity.

Response:
We appreciate the reviewer's insightful comments.We conducted isothermal titration calorimetry (ITC) experiments to determine the binding affinity of NAD + to both DSR2 WT and H171A mutant (refer to the figure below).Surprisingly, no significant binding was detected for either WT DSR2 or H171A mutant.Considering that our new structure of DSR2-tail tube protein (TTP)-NAD + complex revealed the presence of four NAD + densities, whereas the DSR2 H171A structure showed no additional NAD + density, it appears that DSR2 may not exhibit strong binding to the NAD + substrate in the absence of an activator protein.Consequently, we have revised the sentence to convey the following: "This observation suggests that in the absence of an activator protein, DSR2 may lose its ability to bind NAD + ." 2. Then sentence "The HTH domain folds into two HTH motifs and a β hairpin between.' is incomplete.

Response:
We appreciate the reviewer for pointing out our language error.We have rephrased the sentence "The HTH domain folds into two HTH motifs and a β hairpin between" to "The HTH domain folds into two HTH motifs with a β hairpin located between them".
3. Structural comparison of DSR2 and ThsA, Y134 and D135 are supposed to be important for NAD coordination, this conclusion needs to be proved by the biochemical experiments.
Response: This ia a good point.We performed NADase assays to analyze the enzymatic activity of DSR2 Y134A, D135A, and H171A mutants.The results demonstrated that mutations of these key residues, which are conserved in ThsA, obviously reduced the activity of DSR2 (Updated Fig. 1j).
4. In the provided PDB (6LHX), the structure is the apo form ThsA, it is hard to identified the residues for NAD binding.The structure of NAD bound Sir2 in the Sir2-APAZ/pAgo was reported (PMID: 38200015), maybe the comparison of Sir2 in DSR2 and pAgo would be helpful.
Response: This is a great suggestion.During the revision process, the cryo-EM structure of ThsA in complex with NAD + (PDB ID: 8BTP) was also been solved.Consequently, we incorporated the structural comparison of the DSR2 Sir2 domain with the Sir2 domains in ThsA (PDB ID: 8BTP) and Sir2-APAZ/pAgo (PDB ID: 8UAF) complex into the updated version of the manuscript (Updated Figs.1g-i).
5. In figure 2f and extended figure 1a, the results of gel filtration profile of DSR2 are inconsistent.
The peak is 9 ml and 50 ml, please explain.Besides, it is better to use Analytical ultracentrifugation to analysis the effect of the mutation on its oligomerization.Why R86E and Y260E (they are both in the interface 4 ) are eluted differently?
Response: We sincerely appreciate the reviewer's excellent suggestions.The discrepancy in the gel filtration profiles of DSR2 presented in original Fig. 2f (Updated Supplementary Fig. 4a) and original Supplementary Fig. 1a (Updated Supplementary Fig. 1a) is due to the utilization of different size of columns.Specifically, the gel filtration profile shown in original Fig. 2f was obtained using the Superdex™ 200 Increase size exclusion column with 24 mL resin volume, while the gel filtration profile in original Supplementary Fig. 1a was generated using the UNIONDEX 200PG 16/60 size extrusion column with 120 mL resin volume.We have now included this column information in the figure legends for clarity.
In addition, we conducted analytical ultracentrifugation (AUC) experiments to investigate the oligomerization state of WT DSR2 and its mutant proteins.The results demonstrated that DSR2 WT exists as a tetramer in solution, while the DSR2 Y260E mutant forms a dimer, confirming the findings from our previous gel filtration data.However, the AUC profile of DSR2 R86E exhibited a broad peak, and the calculated molecular weight was close to that of a monomer (130 kD).
Additionally, the enzymatic activity assay revealed that the R86E mutation dramatically reduced the activity of DSR2 (refer to the figure below).These findings indicate that the R86E mutation likely impacts the folding of DSR2.Considering that the DSR2 Y260E mutant, which aligns with our expectations and exhibits similar activity to the DSR2 WT protein, we have decided to exclude the gel filtration analysis results for the DSR2 R86E mutant in the revised manuscript.6.The author state that the oligomeric DSR2 lacks NAD hydrolysis activity, so why they used DSR2 H171A mutant to collect the cryo-EM data?If the DSR2 alone is inactivated, how the DSAD1 inhibits its NADase activity?Maybe DSAD1 and the phage tail protein (the activator) competitive bind DSR2?So, the author needs to test the binding affinity of DSR2 and DASD1, and phage tail.

Response:
We thank the reviewer for these great suggestions.In the previous version of our manuscript, we were unable to conduct the NAD + hydrolysis assay, making it unclear whether the DSR2 tetramer alone is active in vitro.Therefore, we utilized the DSR2 H171A mutant to collect data.However, in the revised version, we not only successfully performed the NAD + hydrolysis assay but also determined the structure of the DSR2-TTP-NAD + complex.The NADase assay demonstrated that DSR2 alone is inactive but can be activated by TTP.Additionally, the enzymatic activity of DSR2 triggered by TTP can be suppressed by DSAD1, irrespective of whether DSR2 encounters TTP or DSAD1 first (Updated Figs.6a-b).Our new structure revealed that TTP binds to the same pocket on DSR2 as DSAD1, suggesting competitive binding between DSAD1 and TTP (Updated Fig. 3).Although we attempted to measure the binding affinities of DSR2 to DSAD1 and TTP using isothermal titration calorimetry (ITC), both TTP and DSAD1 proteins exhibited a tendency to aggregate, leading to inconclusive results.As an alternative approach, we conducted a pull-down assay to compare the binding affinity of TTP and DSAD1 to DSR2.The results clearly demonstrated that DSAD1 effectively inhibits the binding of TTP to DSR2, even when DSR2 encounters TTP first (Updated Fig. 6c).Overall, our structural and biochemical analyses provide compelling evidence that DSAD1 competes with TTP for binding to DSR2 and subsequently suppresses its enzymatic activity.
Reviewer #3 (Remarks to the Author): Bacteria and archaea use an expanded set of antiviral defenses to protect against infection by phage.One such defense system, DSR2, uses an NAD+ depletion strategy through the activity of a SIR2 domain.In this manuscript, Wang and Xu et al. use cryo-EM to determine the 3D structure of a tetrameric DSR2 complex providing the first structural information regarding this defense system.
The authors also reveal the structure of DSR2 in complex with the virally encoded anti-defense protein DSAD1 which was previously shown to inhibit DSR2 enzymatic activity.These structures suggest a mechanism for anti-defense by DSAD1 which likely locks DSR2 in a conformation which is not conducive to NAD+ binding and catalysis.
The results of this manuscript are clearly presented.The structural information provided in this manuscript adds to our understanding of NAD+ depleting prokaryotic defense systems which operate through oligomerization.The manuscript, while well organized, lacks data which would shore up some of the conclusions and make it overall a more comprehensive story.Importantly, NADase assays are absent in the current study but would be essential to explain how the oligomers observed in cryo-EM maps may be operating.
Response: we thank the reviewer for the cogent summary of our work and overall positive assessment of our work.

Suggested revisions:
1. Missing word in introduction-"While in bacteria, Sir2 domain proteins recently identified to act as NADases to hydrolyze the bacterial cellular NAD+ to against phage infection."-Missing a word between to and against.Perhaps "protect"?
Response: We appreciate the reviewer for bringing this language error to our attention.We have now incorporated the word "protect" into the sentence.
2. The fact that NAD+ was not observed in the binding pocket of DSR2 may be due to the mutation but it is more than likely that NAD+ cannot bind until DSR2 is conformationally triggered by phage tail tube protein.This alternative interpretation comes in some form much later in the discussion but may be worth stating much earlier.
Response: This is a great suggestion.Based on our new biochemical and structural results, we have incorporated the sentence "This observation suggests that in the absence of an activator protein, DSR2 may lose its ability to bind NAD + " into the second paragraph of the Results section in the updated manuscript.Response: This is a good point.We have performed analytical ultracentrifugation (AUC) experiments to accurately measure the molecular weights of WT DSR2 and its mutants.The results demonstrated that WT DSR2 exists as a tetramer in solution, while the DSR2 Y260E mutant forms a dimer, which is highly consistent with the previous gel filtration data.However, the AUC profile of DSR2 R86E exhibited a broad peak, and the calculated molecular weight was close to that of a monomer (130 kD).Additionally, the enzymatic activity assay revealed that the R86E mutation dramatically reduced the activity of DSR2 (refer to the figure below).These findings indicate that the R86E mutation is likely to disrupt the folding of DSR2.Considering that the DSR2 Y260E mutant, which aligns with our expectations and exhibits similar activity to WT DSR2, we have decided to exclude the gel filtration analysis results for the DSR2 R86E mutant in the revised manuscript. 5.While the subject of the manuscript is the inactivation mechanism of DSAD1 on DSR2 as determined by cryo-EM, there is no NADase data presented showing any inactivation.It might be beneficial to understanding the mechanism if some NADase measurements could be made.At minimum, the impact of tetramer and dimer interface mutants on NADase activity should be explored to highlight the importance of these complexes on activity but also the impact of DSAD1 mutants on NADase inactivation could be explored to further emphasize the importance of these interactions.

Response:
We appreciate the reviewer's insightful suggestion.In the revised manuscript, we carried out the NAD + hydrolase assay to assess the inhibitory capacity of WT DSAD1 or its mutants on the enzymatic activity of DSR2 in the presence of TTP.The activity assay revealed that DSAD1 mutants, which exhibit defects in binding to DSR2, abolished the inhibition of DSR2 NADase activity triggered by TTP (Updated Fig. 5f).Additionally, we analyzed the NADase activity of DSR2 mutants that disrupt the dimer interfaces (L1000A/M1001A and N202A) or the tetramer interface (Y260E) of DSR2 (Updated Fig. 2f).The results indicated that the dimerization of DSR2 is crucial for its enzymatic activity, while the tetramer interface is dispensable for its activation.
6. Unless the authors can show it directly using mutagenesis, binding affinity measurements, and/or cryo-EM, the references to competition of binding to DSR2 by DSAD1 and tail tube should be removed.The model as it stands is incomplete without evidence of direct competition.The analysis should reflect this uncertainty-DSAD1 may bind to a completely separate site relative to the binding site for tail tube and therefore binding would not be considered competitive.For example, there is no evidence to suggest that DSAD1 dislodges the tail tube as seemingly presented in Fig. 5d.

Response:
We thank the reviewer for these constructive comments.During the revision process, we made remarkable progress in this project by successfully determining the cryo-EM structure of the DSR2-TTP-NAD + complex (Updated Fig. 3).This new structure unveils that TTP binds to the identical pocket on DSR2 as DSAD1, suggesting a competitive binding interaction between DSAD1 and TTP to DSR2.To confirm the competitive binding mode, we conducted the NADase assay with varying orders of DSAD1 and TTP addition to DSR2.The activity studies demonstrated that DSAD1 effectively suppresses the enzymatic activity of DSR2 triggered by TTP, irrespective of whether DSR2 encounters TTP or DSAD1 first (Updated Figs.6a-b).However, TTP can not obviously activate DSR2 in the presence of DSAD1.In addition, the subsequent pull-down assay revealed that DSAD1 significantly inhibits the binding of TTP to DSR2, even when DSR2 is preincubated with TTP (Updated Fig. 6c).In all, our structural and biochemical findings support two models.Firstly, if the DSAD1 protein is expressed first upon bacterial infection by phages, DSAD1 binds to DSR2 and locks it in an inactive state, preventing abortive infection.On the other hand, if TTP is expressed before DSAD1 and forms the DSR2-TTP complex, DSAD1 displaces TTP from DSR2, leading to the inhibition of its enzymatic activity.As we do not have information regarding which protein is expressed first after infection, we have revised our model by excluding the possibility of DSAD1 displacing TTP from DSR2.
Wang et al. report on the structural basis of inhibition of the Dsr2 defence protein by the phageencoded DSAD1 protein.Structures of DSR2 and the complex with DSAD1 using cryoEM are presented, and conclusions tested by sdm.

a,
Gel filtration profiles of DSR2 WT and R86E, Y260E mutant proteins on the Superdex™ 200 Increase size exclusion column.b, Analytical ultracentrifugation analysis the molecular weight of WT DSR2 and its R86E, Y260E mutant proteins.c, NAD + hydrolase activities of WT DSR2 and its mutants in the presence of TTP.
3. Typo TshA -> ThsA "To elucidate the mechanism of NAD+ binding, we conducted a detailed analysis of the overlaid structures of DSR2 and TshA."Response:We thank the reviewer for pointing out this typo in the manuscript which has been corrected.4.Can the authors more accurately/quantitatively describe the oligomeric state of the tetramer interface mutants?"The gel filtration profiles of the DSR2 mutant proteins exhibited slower migration compared to the wild-type protein, indicating disruption of DSR2 tetramerization (Fig.2f)."If molecular weight standards were run this could indicate if the shifted peaks are representative of monomer or dimeric complexes.

a,
Gel filtration profiles of DSR2 WT and R86E, Y260E mutant proteins on the Superdex™ 200 Increase size exclusion column.b, Analytical ultracentrifugation analysis the molecular weight of WT DSR2 and its R86E, Y260E mutant proteins.c, NAD + hydrolase activities of WT DSR2 or its mutants in the presence of TTP.